Process for synthesis of silyl alcohols

ABSTRACT

Novel silyl alcohols having bulky substituents bonded to the silicon, and the silyl group attached to a carbon include the preferred 2-silyl-ethan-1-ols. A method for synthesizing silyl substituted alcohols include hydrosilation of a vinylic ester, especially vinyl acetate, followed by hydrolysis in mild base. The silyl alcohols are useful in preparing phosphorylating reagents for phosphorylating an oligonucleotide. The phosphorylated intermediate bearing the silyl group may be separated from failure product on the basis of bulky substituents on the silyl protecting group, which is later removed, e.g. by fluoride ion.

The invention relates generally to silyl alcohols, their synthesis, andtheir use. More specifically, the invention relates to specific silylalcohols and to a method for synthesizing silyl alcohols having thesilicon atom bonded to a carbon rather than to the hydroxyl. Theinvention also relates to reagents and methods for phosphorylatingoligonucleotides, and to intermediate compounds and methods useful forpurification of phosphorylated oligonucleotides.

This application is related to co-owned and co-pending applications Ser.Nos. 07/712,001, filed Jun. 7, 1991 and 07/712,302 filed Jun. 7, 1991,each of which is incorporated herein by reference.

BACKGROUND

Chemically synthesized oligonucleotides have been used in hybridizationassays for some time, and by now are fairly routine. However, for useswhich imitate biological processes, e.g. hybridizations of nucleic acidprobes on a template followed by ligation, the normal 5' hydroxylterminus must be converted to a phosphate to provide the propersubstrate for a ligase. Methods of phosphorylating include enzymatic andsynthetic as described below. The present invention describes aparticular synthetic method, wherein silyl substituted alcohols areuseful reagents.

Synthesis of silyl substituted alcohols has been previously achieved byoxidation of organoboranes. The organoboranes are in turn prepared bythe Grignard reaction or by hydroboration of vinyl- and allyl-silanes.This technique is described in Kumada, et al. J. Organometal. Chem. 6:490-495 (1966) and Seyferth, J. Am. Chem. Soc. 81: 1844 (1959). Thistechnique is useful only when the requisite vinyl or allylsilanes can besynthesized or obtained commercially. However, if the desired vinylsilane is commercially unavailable or difficult to synthesize thismethod is not useful.

Alpha silyl esters have been prepared by reacting a chlorosilane and analpha-bromo ester with zinc under Reformatsky conditions. See Fessenden,et al., J. Org. Chem. 32: 3535 (1967).

An important drawback of these synthesis methods is the side reactionswhich can occur leading to undesirable products and decreasing theyields. In conventional processes for hydrolyzing silyl substitutedesters to the corresponding silyl substituted alcohol, a carbanionintermediate is generally formed. With β-silyl substituted alcohols,fragmentation to the silanol and an olefin can occur; with alpha silylsubstituted alcohols, a Brook rearrangement to give a silyl protectedether will occur. Thus, in these carbanion intermediates there is astrong tendency for an elimination reaction whereby the silicon atomshifts to the oxygen atom to form the R₃ SiOH byproduct. This tendencyis especially pronounced when the reaction is performed in strong baseand when groups substituted on the silicon are particularly bulky.

Hydrosilation, the addition of H and silyl compounds across the doublebond of an olefin, has also been described in the literature. SeeCollman, et al. Principles and Applications of Organotransition MetalChemistry, University Science Books (1980) p. 384-389 and Pegram, et al.Carbohydrate Research 184: 276 (1988). In a particularly relevanthydrosilation reaction, Salimgareeva, et al., Zh. Obshch. Khim 48(4):930-31 (1978)(Russian) (see also C.A. 89: 146961y) report hydrosilationof vinyl acetate with dimethylsilane. This reaction resulted in twosilyl substituted products: a monoacetate and a diacetate. The referencefails to describe synthesis of any silyl alcohol or the use or synthesisof any bulky silyl substituted compound.

Honda, et al. Tetrahedron Letters, 22(22): 2093-2096 (1981) describe aβ-silyl substituted ethanol wherein the silyl group bears two phenyl andone methyl substituent. Honda, et al. used this compound to prepare aphosphorylating agent which places a protected phosphate group betweennucleotides in oligonucleotide synthesis. The substituted silylprotecting group can be removed to give a silyl fluoride compound,ethylene and the phosphate. The substituted silyl ethanol was obtainedby reduction of the bisphenylmethyl silyl acetate with LiAlH₄ accordingto a modification of the procedure of Gerlach, Helv Chim. Acta, 60: 3039(1977).

Other silyl substituted ethanols have been described in the literature,but primarily include alkyl substituted silyl groups. Examples of suchsilyl ethanols and their literature citations are found in the followingtable.

                  TABLE 1                                                         ______________________________________                                         ##STR1##                                                                     R      R'       R"       Literature Citation                                  ______________________________________                                        isopropyl                                                                            isopropyl                                                                              isopropyl                                                                              CA111(11):97352n                                     methyl methyl   propenyl CA105(13):115112s                                                             CA108(17):150554w                                    methyl n-butyl  n-butyl  CA88(3)23391j:                                                                CA83(11):97563k;                                                              CA78(15):93640g; &                                                            CA78(13):84526x                                      methyl methyl   t-butyl  CAOLD (prior to 1967)                                ethyl  ethyl    ethyl    CA111(11):973552n;                                                            CA98(9):72207u;                                                               CA87(15):117488c;                                                             CA85(21):154709e;                                                             CA80(11):59132z; &                                                            CA77(18):120049a                                     propyl propyl   propyl   CA103(19):160573n                                    phenyl phenyl   methyl   Honda, et al., Tetrahedron                                                    Letters, 22(22):2093-2096 (1981)                     ______________________________________                                    

Triphenyl silane (not the alcohol) has been described by Lesage, et al.J. Org. Chem 55: 5413 (1990) as a useful reducing agent.

In addition to the method of Honda, et al. (See above), several methodsfor phosphorylating the 5' terminus of an oligonucleotide are known.Initially, enzymatic methods using polynucleotide kinase were employedafter the oligonucleotide was synthesized and removed from the solidsupport. Others have taught methods and reagents for chemicallyphosphorylating a synthesized oligonucleotide prior to its removal fromthe solid support. Some of these are described below.

Kondo, et al. Nucl. Acids Res. Symposium Series 16: 161-164 (1985)describe phosphotriester (1) and phosphoramidite (2) reagents forphosphorylating 5' termini. Phosphorylation is achieved by preparing aspecial diphosphorylated (3'-5') nucleotide which is added as the lastnucleotide in the chain. The 3' phosphate is linked via thephosphotriester or phosphoramidite to the extending nucleotide chain.The 5' phosphate is protected with a protecting group which isultimately removed.

Uhlmann, et al. Tetrahedron Letters 27(9): 1023-1026 (1986) describe aphosphoramidite phosphorylating reagent using a p-nitrophenylethyl groupas a blocking group. They mention that the hydrophobicp-nitrophenylethyl is advantageous in that phosphorylated compounds canbe separated from non-phosphorylated compounds by reversed phase HPLC.

Uhlmann, et al, however, used only hexamers to which thep-nitrophenylethyl "handle" was attached. A similar approach using ap-nitrophenylethyl handle with 20-mers is described by G. Zon in chapter14 of HPLC in Biotechnology, (W. S. Hancock, ed), J. Wiley & Sons, NewYork, N.Y. pp 359-363 (1990). The purification results obtained by Zonwith this method are marginal.

Marugg, et al. Nucl. Acids Res. 12(22): 8639-8651 (1984) describe a newphosphorylating agent, 2-cyano-1,1-dimethylethoxy dichlorophosphine.This agent has the alleged advantage of being removed under just basicconditions.

Himmelsbach, et al. Tetrahedron Letters 23(46): 4793-4796 (1982)describe a new phosphorylating agent, bis-(p-nitrophenylethyl)phosphoromonochloridate. Van der Marel, et al. Tetrahedron Letters,22(19): 1463-1466 (1981) describe a morpholino phosphoro bis-3-nitro-1,2, 4-triazolidate.

Horn, et al. Tetrahedron Letters 27 (39): 4705-4708 (1986) describe aphosphorylating reagent including a 4, 4' dimethoxytrityl group which,upon release, can be used to monitor the efficiency of phosphorylation.This disclosure appears to be quite similar to that of EP-A-304 215 andto the commercially available Clontech product known as 5' Phosphate-On.

Lipshutz, et al. Tetrahedron Letters 30(51): 7149-7152 (1989) ("Lipshutz1989") and Lipshutz, et al. Tetrahedron Letters 21: 3343-3346 (1980)("Lipschutz 1980") and Von Peter Sieber, Helvetica Chimica Acta 60: 2711(1977) all disclose the use of fluoride in the removal of a silylprotecting group. In this regard, they are similar to Honda, et al. (Seeabove).

While each of the above reagents and methods are adequate forphosphorylating synthesized oligonucleotides, each has draw backs aswell. For example, each of the recited references discloses a method forremoving the phosphate blocking group to generate the native 5'phosphate. Some (e.g. Horn, et al.) describe a blocking agent having adetectable characteristic (e.g. color) by which the extent ofphosphorylation can be monitored. While the extent of phosphorylationcan be monitored by this means, it provides no means for purification.Uhlmann, et al. suggest that the hydrophobic p-nitrophenylethyl groupcan be used prior to cleavage to separate phosphorylated hexamers byHPLC. The protected hexamers cited by Uhlmann, having a relatively lowmolecule/protecting group mass ratio, are generally too short to providespecificity necessary in hybridization assays.

However, none of the references teach phosphorylating/blocking reagentscomprising silyl substitutes. Further, none suggest that the silylprotecting group can be used to purify phosphorylated nucleotides fromunphosphorylated failure product. The present invention seeks toovercome these disadvantages.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a process for synthesis ofsilyl alcohols, comprising:

a) hydrosilation of an vinylic ester of the formula ##STR2## whereinR_(a), is a straight or branched alkenyl group having from 2 to about 20carbons and R_(b) is H or lower alkyl, with a silane of the formula##STR3## wherein R₅, R₆ and R₇ are independently selected from the groupconsisting of H, alkyl, aryl, substituted alkyl, substituted aryl, oxaand thia analogs of alkyl, aryl, substituted alkyl and substituted aryl,and halogen, in the presence of a metal catalyst capable of catalyzingthe addition of H and a silyl group across the double bond of thealkenyl group to form a silyl substituted ester; and

b) hydrolyzing the silyl substituted ester to an alcohol in the presenceof mild base.

Generally, R_(a) is lower alkenyl; preferably R_(a) is vinyl. Generally,R_(b) is lower alkyl; preferably methyl. For use as a phosphorylatingreagent, at least one of R₅, R₆ and R₇, preferably more than one, shouldconsist of a sterically bulky, and ideally a hydrophobic, group.

Preferably, hydrosilation is done in a solution of toluene ranging fromabout 0.1 to about 4 molar, especially about 1 molar. The metal catalystis usually a transition metal complex having a metal selected from thegroup consisting of cobalt, nickel, platinum, palladium and rhodium, forexample, {RhCl(CO)₂ }₂. Hydrolysis is usually done in a protic solventsuch as methanol or ethanol with a mild base such as K₂ CO₃.

The process may further comprise a step of separating the silylsubstituted alcohol from undesired products of side reactions, forexample, by chromatography.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chromatogram showing separation of a phosphorylatedoligonucleotide (peak 4 at 15.5 min) from the failure products (peak 1at 8.4 min). The chromatogram was generated from a Waters μBondapak™ C18column, 3.9 mm×150 mm flowing at 1.5 mL/min. Solvent A was 100 mMTriethylammonium Acetate and solvent B was Acetonitrile. Solvents weremixed according to a linear gradient table such that the ratio of A:Bwas as follows: At time=0, 90:10; at time=15 min, 60:40; at time=25,60:40; and at time=30, 90:10. Detection was in absorbance units at 260nm. (See Example 9a.)

FIG. 2 is a chromatogram showing separation of deprotected,phosphorylated oligonucleotide (peak 1 at 8.4 min) from other products(e.g. silylfluorides). The conditions are the same as in FIG. 1. (SeeExample 9b.)

DETAILED DESCRIPTION A. General Definitions

In general, terms like "alkyl", "alkenyl" and "aryl" have the meaningsusually attributed to them by persons skilled in the art of organicchemistry. For example, alkyl refers generally to monovalent straight orbranched aliphatic radicals which may be derived from alkanes by theremoval of one hydrogen, and have the general formula C_(n) H_(2n+1).Alkyl groups may have from 1 to about 30 carbons, more practically 1 toabout 15 or 20. "Lower alkyl" refers to alkyls having from 1 to about 6carbons. Examples of lower alkyl include CH₃ --, CH₃ CH₂ --, CH₃CH(CH₃)--, and CH₃ (CH₂)₄ --. As used herein, "alkyl" includescycloalkyl as well as straight alkyl. Thus, cyclohexyl and others areincluded.

"Alkenyl" refers to monovalent straight or branched aliphatic radicalswhich may be derived from alkenes by the removal of one hydrogen, andhave the general formula C_(n) H_(2n-1). Alkenyl substituents may havefrom 1 to about 30 carbons, more practically 1 to about 20. "Loweralkenyl" refers to alkenyls having from 1 to about 6 carbons. "Olefinic"is a synonym for alkenyl.

As used herein, "alkylene" refers to a divalent straight or branchedchain spacer group containing less than 30 carbon atoms, including butnot limited to, --CH₂ --, --CH(CH₃)--, --CH(C₂ H₅)--, --CH(CH₃)CH₂ --,--(CH₂)₃ --, and the like. Generally, an alkylene spacer group isaliphatic.

"Aryl" refers to a monovalent radical derived from aromatic hydrocarbonsby the removal of one hydrogen. Aryl substituents having ringstructures, such as those of phenyl and naphthyl. Typically, arylsubstituents are planar with the π electron clouds of each carbonremaining on opposite sides of the plane.

Although alkyl, alkenyl and aryl are generally limited to groups havingno atoms other than carbon and hydrogen (i.e. no heteroatoms), theinvention is not so limited. Heteroatoms, especially oxygen and sulfur,can be present in "R" groups to form "oxa" and "thia" analogs,respectively. However, because of the anticipated elimination, it isdesirable to avoid oxa analogs having an oxygen atom 2 carbons removedfrom the point of monovalency where the R group is attached to themolecule of interest. Exemplary oxa analogs include alkoxy, such ast-butoxy, isopropyloxy and ethoxy, phenoxy and ether substituents.

As used herein, "substituted" refers to the presence of moietiescovalently bonded to the "R" groups, including, but not limited to,halide (especially Br and Cl), nitro, lower alkoxy (having from 1-6carbon atoms, especially methoxy and ethoxy), lower alkyl (having from1-6 carbon atoms, especially methyl and ethyl), hydroxy, and amino(protecting group may be required). Subject to constraints imposed bythe desired solubility, and hydrophobicity of the desired compound, andby the steric constraints or organic chemistry principles, thesubstituting groups may be placed anywhere, and in any number, on the Rgroup. Some specific substitutions include: Alkaryl, which refers to amonovalent aryl radical bearing alkyl substituents where the arylradical includes the point of monovalency (e.g. toluyl); and Aralkyl,which refers to monovalent alkyl radicals bearing aryl substituents. Inthis latter case, the alkyl radical includes the point of monovalency.Benzyl is an example of an aralkyl group.

As used herein, "sterically bulky" refers to substituents groups whichoccupy a relatively large volume. Aryl groups having five or morecarbons are considered "sterically bulky", as are substituted arylgroups. Alkyl and alkenyl groups are "sterically bulky" when theypossess at least 4 carbons and are arranged in a branched configuration,the more branches, the bulkier. Any alkyl occupying a volume equal to orlarger than t-butyl; and any aryl occupying a volume equal to or largerthan phenyl, is considered "sterically bulky". Thus, neopentyl, neohexyland others meet this description.

"Hydrophobic" refers generally to compounds which are relativelyinsoluble in aqueous solutions and will not substantially mix withwater. Specifically, a compound is deemed hydrophobic if it has apartition coefficient of 0.51 or greater to octanol in a water/octanolpartitioning test.

B. Silyl Alcohol Synthesis

Silyl alcohols prepared by any method may be useful in the inventionsdescribed below. 2-silyl-ethan-1-ols (or β-silylethanols orsilapropanols) are preferred for reasons which will become apparent. Itwill be readily apparent to those of ordinary skill in the organicchemistry arts that the terms "β-silylethanol" and "silapropanol" areequivalent and may be used interchangeably. The former method ofnomenclature treats the silyl group (R₃ Si--) as a substituent on theethanol, while the later method treats the silicon atom as part of thebackbone.

Some of the known preparation methods are set forth in the Background ofthis application. However, one novel method of synthesis is particularlyuseful and is described here.

Vinylic esters are olefinic esters characterized by the presence of thealkenyl group on one side (the oxygen side) of the ester linkage. Theymay be represented by the formula: ##STR4## wherein R_(a) is alkenyl andR_(b) is H or alkyl, usually lower alkyl, and preferably methyl. In thisinvention, R_(a) may be from 2 to about 30 carbons, but more commonly islower alkenyl. Examples of such esters useful in the invention includevinyl acetate, isopropenyl acetate, butenyl acetate, pentenyl acetate,and etc. Esters wherein the double bond is in the terminal position arepreferred, especially vinyl acetate.

Hydrosilation of such esters using a silane of the formula R₃ SiH in thepresence of a metal catalyst adds H and a silyl group (R₃ Si--) acrossthe double bond of the alkenyl group R_(a). Hydrosilation requires asilane, preferably bearing alkyl, aryl, substituted alkyl, orsubstituted aryl as the "R" groups. Any "R" group of the silane may alsoindependently include halogen and/or oxa or thia analogs of alkyl, aryland substituted alkyl or aryl. There may be one, two or three "R" groupson the silane. For the uses described below, bulky, hydrophobicsubstituents are preferred. Phenyl, t-butyl, neopentyl, etc, areexemplary bulky groups.

Metal catalysts useful for hydrosilation include transition-metalcomplexes, particularly those of cobalt, nickel, platinum, palladium andrhodium, although others may work as well. Specific complexes includeCo₂ (CO)₈ ; H₂ PtCl₆ ; {RhCl(CO)₂ }₂ ; and others given in Table 6.5 ofCollman et al., supra.

Catalytic hydrosilation can be performed under the following conditions.The molar ratio of acetate to silane can range from about 30:1 to about1:2, and is preferably about 1:1. Intermediate ratios, such as 10:1 or2:1, are contemplated as well. The metal catalyst may be present in molepercentages ranging from about 0.01% to about 3%, preferably betweenabout 0.2% and about 2%. Lower percentages may require longer reactiontimes or higher temperatures. For {RhCl(CO)₂ }₂ an optimal mole % isbetween about 0.25% and about 1.0%. For other catalysts, the optimalconcentrations can be obtained from the literature or from routineexperimentation. The reaction is best run at room temperature for about50-70 hours, preferably not longer than 2 weeks. It may, however,proceed more quickly at elevated temperatures; for example, in less than24 hours at 82° C. The principle reagents should be present at aconcentration ranging from neat in vinyl acetate to 4M in toluene;preferably about 1M in toluene. Other reaction conditions for thiscatalytic step can be found in Collman, et al., which is incorporated byreference.

In hydrosilation, two major products result because the silylsubstituent may bond to either side of the double bond to give both 1-and 2- substituted products. If necessary, these can be separated andpurified by chromatography, for example, silica based chromatographysuch as flash column or HPLC. However, in a specific instance,purification is greatly simplified. When vinyl acetate is used as theester, two products are again obtained as follows: ##STR5##

Upon hydrolysis in mild aqueous or alcoholic base, the acetate isconverted to an alcohol. However, the 1-silyl substituted alcohol isunstable and spontaneously undergoes a Brook rearrangement (A. G. Brook,Accounts Chemical Research, 7:77 (1974)) to give: ##STR6## The 2-silylsubstituted alcohol does not undergo this rearrangement. Since the2-silyl substituted product behaves as an alcohol, while the silyl etherbehaves as an ether, the two products are easily separated on the basisof these properties using silica gel chromatography, especially HPLC. Aswill be seen from the examples which follow, this hydrolysis reactioncan be run in the same vessel without any intermediate purification ofthe acetate.

The conditions of hydrolysis preferably are carefully controlled. Itwill be recalled that 2- or β- substituted intermediates in the anionicform will undergo fragmentation to the silanol as described in theBackground section. However, reaction conditions can be selected whichwill minimize the formation of the undesired product. First, a mild baseis selected, preferably one having a pK_(b) between about 3 and 8.Acceptable bases include the sodium or potassium salts of H₂ BO₃ -, HPO₄²⁻, SO₃ ²⁻, HCO₃ - and CO₃ ²⁻. A mild base, having only a weak tendencyto dissociate, tends to keep anionic species protonated moreso thanstrong bases or hydrides (e.g. LiAlH₄) taught in the prior art.

The base should be present in a base:acetate molar ratio of from about0.01:1 to about 3:1, preferably between 0.1:1 and 2.5:1, most preferablybetween 1:1 and 2:1. The reaction generally takes from 0.5 to 24 h, butpreferably takes about 1 hour. In addition, solvents can be selected tominimize the formation of undesired product. For example, the solubilityof the base in a particular solvent will affect its strength. It isdesirable to have a substantial amount of the base insoluble so as tobuffer the ionization equilibrium going on in solution. Also, proticsolvents are preferred over aprotic solvents, due to their ability toquench the formation of anionic species. Suitable protic solventsinclude water, methanol and ethanol. Although the reaction will work inan aqueous medium, it is preferable to use methanol as the solvent.

Silyl alcohols synthesized by this method, as well as silyl alcoholssynthesized by other processes, find utility in the synthesis ofphosphorylating reagents and protecting agents as described in a latersection.

C. Silyl Alcohols

While the above-described method may be used to synthesize many silylsubstituted alcohols, one class of silyl alcohols is of particularinterest. Previous methods have not been known to synthesize silylalcohols having three large, bulky groups bonded to the silicon. This isbecause the principal prior art method of synthesis--i.e., via vinylsilanes as taught by Kumada, et al and Seyferth, supra--requires vinylsilane reagents appropriately substituted with the necessary bulkygroups. Presumably due to steric considerations, bulky vinyl silanes arenot readily available or easily synthesized. Although triphenyl silaneis known (see Lesage, et al. supra), such a bulky silane has not beenassociated with a vinyl radical to make the bulky vinyl silane.

However, sterically bulky silyl alcohols can be made by the abovedescribed method, and have the general formula: ##STR7## wherein R₁, R₂and R₃ are independently selected from sterically bulky groups like aryl(e.g. phenyl and naphthyl), substituted aryl (e.g. methoxyphenyl, ornitrophenyl) aralkyl (e.g. triphenylmethyl), alkaryl and alkyl orsubstituted alkyl having at least 4 carbons in a branched chain (e.g.t-butyl, neopentyl, neohexyl, cyclohexyl, 3-pentyl and3-ethyl-3-pentyl). In the formula above, n is an integer from 2 to about20, usually 2 to about 6 and most preferably 2. Exemplary compounds arelisted in the table below, although this is by no means an exhaustivelist.

                  TABLE 2                                                         ______________________________________                                        Illustrative Novel Silyl Alcohols                                             R.sub.1     R.sub.2     R.sub.3   n                                           ______________________________________                                        phenyl      phenyl      phenyl    2                                           phenyl      phenyl      phenyl    6                                           phenyl      phenyl      t-butyl   2                                           phenyl      t-butyl     t-butyl   2                                           phenyl      naphthyl    neopentyl 2                                           t-butyl     t-butyl     neopentyl 2                                           phenyl      naphthyl    t-butyl   2                                           phenyl      t-butyl     neohexyl  2                                           phenyl      phenyl      phenyl    3                                           phenyl      phenyl      t-butyl   3                                           t-butyl     t-butyl     phenyl    3                                           phenyl      naphthyl    neopentyl 3                                           t-butyl     t-butyl     neopentyl 3                                           phenyl      naphthyl    t-butyl   3                                           phenyl      t-butyl     neohexyl  3                                           ______________________________________                                    

For reasons which will become apparent, substituted bulky groupspreferably are substituted with nonpolar substituents.

As mentioned, silyl alcohols find utility in preparing phosphorylatingagents and protecting agents. These are described in detail below.

D. Phosphorylating Reagents

Many types of reagents can phosphorylate--i.e. put a phosphate group onthe end of--an oligonucleotide. Generally these reagents are classifiedas phosphotriester reagents, phosphonate reagents (Hydrogen or Alkyl)and phosphoramidite reagents. The mechanisms by which each of thesereagents phosphorylate an oligonucleotide is described in theliterature.

A novel phosphorylating reagent is represented by the formula: ##STR8##wherein R₅, R₆ and R₇ are independently selected from H, alkyl, aryl,substituted alkyl, substituted aryl, oxa and thia analogs of alkyl,aryl, substituted alkyl and substituted aryl, and halogen; and wherein Qrepresents a moiety selected from the group consisting ofphosphoramidites, alkyl phosphonates, hydrogen phosphonates andphosphotriesters.

For a phosphoramidite, Q has the formula: ##STR9## and R₈ is generallyselected from the group consisting of 2-cyanoethyl, methyl, ethyl,2-alkylsulfonylethyl, 2-(p-nitrophenyl)ethyl, 2-(9-fluorenyl)ethyl,2-(2-anthraquinonyl)ethyl, 2-alkylthioethyl, 2-arylthioethyl,2-trihalomethylethyl, 2-phenylethyl and 2-(2-naphthyl)ethyl. R₉ and R₁₀are generally selected independently from H, or straight or branchedalkyl having from 1-6 carbons. In a very common phosphoramidite moiety,R₈ is 2-cyanoethyl while R₉ and R₁₀ are both isopropyl.

The novel silyl phosphoramidite may be prepared in a conventional mannerby reacting a chlorophosphoramidite with a silyl substituted alcohol.See, e.g., Koster Tetrahedron Letters, 24:5843 (1983) which isincorporated herein by reference. Here, it is preferred to use a2-silyl-ethan-1-ol. The reaction conditions are well known from theliterature.

For a phosphotriester reagent, Q has the formula: ##STR10##

For hydrogen phosphonate or alkyl phosphonate reagents, Q has the aboveformula, but Y is H or alkyl, respectively.

E. Methods Using Phosphorylating Agents

The above-described phosphoramidite, phosphotriester and phosphonatereagents can be used in a method for phosphorylating an oligonucleotide,particularly an oligonucleotide synthesized on a solid support. It willbe realized by those of ordinary skill in the art that a singlenucleoside could equally well be phosphorylated in this manner, as couldlonger polynucleotides. For simplicity, it will be understood that theterm "oligonucleotide" will include structures having from one toseveral hundred nucleoside subunits.

Many methods are known in the literature for synthesizingoligonucleotides and the particular method employed is not relevant tothe present invention. Generally, however, automated synthesis ispreferred and may be performed using commercial instruments such as anABI 380A Synthesizer or a Milligen 8700 Synthesizer.

The reaction steps employed by such automated synthesizers are generallyknown in the art and need not be repeated here. However, it will benoted that when a phosphoramidite or H phosphonate reagent is used, theresulting intermediate is a trivalent phosphite. It is subsequentlyoxidized to the biologically useful pentavalent phosphate. Thisoxidation step is readily achieved using, for example iodine, in theautomated synthesis process.

A major advantage of the present invention is that the phosphorylationstep can be accomplished in the same instrument as synthesis, withoutremoval of the oligonucleotide from the support. Alternatively,oligonucleotides synthesized by other methods (e.g. enzymatic) may bephosphorylated by the methods of the present invention, provided theamino and hydroxy functions present can be protected.

While known methods of phosphorylating have been described in theBackground section, none use silyl reagents. Any of the phosphorylatingreagents prepared in the preceding section, may be used to phosphorylatean oligonucleotide according to the invention. The methods andconditions are conventional, although the reagents are not. The examplesprovide further details but the method generally comprises reacting the5' hydroxyl of an oligonucleoside with a phosphorylating reagentdescribed above, ultimately to form a phosphodiester protected by thesilyl group.

The silyl protected, phosphorylated intermediate has the structure:##STR11## where R₅, R₆ and R₇ are selected as before; Z is H or OH; andBASE represents one of the nucleic acid bases A, C, G, T or U, oranalogs thereof. The terminal nucleoside may be attached at its 3'carbon to a support (in the case of phosphorylating a single nucleoside)or, more likely, to a string of one or more other nucleosides (to forman oligonucleotide). Generally, such a string of nucleosides will beconnected via phosphodiester linkages, although other linkages arepossible (e.g. alkyl phosphonate neutral probes). Obviously, where Z isH, the nucleoside is a deoxyribonucleoside; where Z is OH, it is aribonucleoside. Analogs of the bases A, C, G, T or U are compoundswhich, when incorporated into an oligonucleotide, will still permitWatson-Crick base pairing with their respective complementary base. Someexemplary base analogs are published in the USPTO Official Gazette at1114 OG 43, which is incorporated herein by reference.

While the silyl protecting group must be removed for biological use(e.g. template guided ligation) the protected intermediate also hasutility. The silyl group, particularly if it is endowed with bulky,hydrophobic substituents R₅, R₆ and R₇, is useful as a "handle" forpurifying and separating phosphorylated oligonucleotides fromunphosphorylated failure product by chromatography, e.g. HPLC. Providedthe R groups are sufficiently hydrophobic, the oligonucleotide bearingthe silyl protecting group is easily differentiable from theunphosphorylated, unprotected oligonucleotide, even when theoligonucleotides approach 50-mer lengths. Of course, shorter lengths arealso easily separated. This goes a step beyond the known tritylprotecting groups which are useful to monitor phosphorylation success,but not to separate or purify product.

If desired, a deprotecting step may follow phosphorylation and/orseparation to yield the 5' terminal phosphate. The deprotecting step isdone by any useful method to yield the desired phosphate. A preferredmethod, especially useful when the silyl substituent is β to the oxygenas above, involves reacting the protected phosphodiester with fluorideion to give the silyl fluoride, ethylene and the terminal phosphate.Tetrabutylammonium fluoride (TBAF) is a useful fluoride ion for removingthe silyl protecting group. This reaction is driven by the release ofethylene when the phosphorylating reagent above is used. See, e.g. Grob,Helv. Chim. Acta, 38:594 (1955). It is for this reason that2-silyl-ethan-1-ols (β silylethanols) are preferred silyl alcoholreagents (they have two carbons between the silicon and the oxygen ofthe phosphodiester, thus permitting the Grob elimination of ethylene).Any other length will not be removed in the deprotection step as easilyas the β silyl-ethanol derivative.

"Protecting" group and "deprotecting" steps refer to the silapropylsubstituent attached to the oxygen of the phosphate. This group may ormay not afford "protection" in the usual sense from subsequent reactionsthat would affect the oxygen atom. However, the term is used as asynonym for "handle" because of the ability to separate phosphorylatedoligonucleotide from unphosphorylated failure product using thesilapropyl group, and because of the subsequent removal of the group togive the desired phosphate.

The inventions herein described will be better understood in view of thefollowing examples which are intended to be illustrative andnon-limiting.

EXAMPLES A. Preparation of Silyl Alcohols EXAMPLE 1 a) Preparation of1,1,1-Triphenyl-3-acetoxy-1-silapropane (3) ##STR12##

A solution of 3.69 mL (40 mmol) of vinyl acetate (1), 10.42 g (40 mmol)of triphenylsilane (2), and 77.8 mg (0.25 mmol) of Rh₂ Cl₂ (CO)₄ in 40mL of toluene was stirred at room temperature under N₂ for a total of 63h. Several runs of the reaction at this scale had unpredictableinduction periods, followed by rapid heat evolution. Scaleup of thisreaction should be done with a cooling bath close at hand. The very darkreaction mixture was treated with 5 g of decolorizing charcoal, and themixture boiled briefly. After cooling, the mixture was filtered througha 1 cm pad of Celite™ with filtrate and washings being collected.Solvent was evaporated and the remaining residue was vacuum dried. Atthis point, the crude material was carried on to the hydrolysis step.NMR analysis showed an α:β ratio of 1:1.57. The following protocol wascarried out for compound identification purposes. A 100 mg sample ofcrude material was flash chromatographed using 4% EtOAc in cyclohexaneon a 25 mm I.D.×150 mm long silica gel column. This afforded 29 mg of(3) after recrystallization from MeOH, mp 67°-68° C.

IR: (CDCl₃, cm⁻¹) 3070 (m), 1728 (vs), 1425 (vs), 1249 (vs); MS:(DCl/NH₃) m/e 364 (M+NH₄);

NMR: (300 MHz, CD₂ Cl₂) ∂7.6-7.3 (m, 15H, phenyl), 4.22 (B₂ of A₂ B₂,2H, CH₂ O), 1.87 (s, 3H, CH₃), 1.86 (A₂ of A₂ B₂, 2H, CH₂ Si);

¹³ C NMR: (75 MHz, CDCl₃) ∂171.1 (C═O), 135.5 (meta), 134 (ipso), 129.7(para), 128 (ortho), 62.1 (CH₂ O), 21 (Me), 14.4 (CH₂ Si).

Elemental Analysis: Calc'd for C₂₂ H₂₂ O₂ Si; C: 76.26; H; 6.40. Found;C: 76.45; H: 6.37.

b) Preparation of 1,1,1-Triphenyl-1-silapropane-3-ol (4) ##STR13##

The crude (3) was dissolved in 100 mL MeOH, and 10.0 g of K₂ CO₃ wasadded all at once. The reaction was complete after 1 h of stirring atroom temperature. The solids were filtered off, and the filtrate wasconcentrated. The concentrated residue was partitioned between 100/100mL H₂ O/EtOAc. After solvent removal from the organic layer, the residuewas vacuum dried. Flash chromatography (18% EtOAc in cyclohexane, R_(f)=0.32) using a 41 mm I.D.×150 mm long silica gel column afforded 3.42 g(28%) of (4). Recrystallization from cyclohexane gave the analyticalsample as a snow-white solid, mp 96°-97° C.

IR: (CDCl₃, cm⁻¹) 3616 (m), 2970 (m), 1429 (vs);

MS: (FAB/DMF-Kl) m/e 343 (M+K);

NMR: (300 MHz, CD₃ OD) ∂7.55-7.3 (m, 15H, phenyl), 3.73 (B₂ of A₂ B₂,2H, CH₂ O), 1.78 (A₂ of A₂ B₂, 2H, CH₂ Si);

¹³ C NMR: (75 MHz, CDCl₃) ∂135.5 (meta), 134.4 (ipso), 129.6 (para), 128(ortho), 59.8 (CH₂ O), 18.7 (CH₂ Si).

Elemental Analysis: Calc'd for C₂₀ H₂₀ OSi.0.2 H₂ O; C:77.98; H: 6.67.Found; C: 77.92; H: 6.62.

EXAMPLE 2 a) Preparation of1,1-Dimethyl-1-phenyl-3-acetoxy-1-silapropane

To a solution of 6.13 mL (40 mmol) of PhMe₂ SiH and 3.69 mL of vinylacetate in 40 mL of toluene was added 61.3 mg (0.16 mmol) of Rh₂ Cl₂(CO)₄. Immediately, the reaction evolved heat and gas. Within 5 min, thegolden yellow reaction had turned dark brown in color. After 1 h, thereaction was complete. The reaction was worked up as in example 1a togive 8.39 g of crude adduct. Proton NMR analysis showed an α:β additionratio of 1.44:1.0. A 100 mg sample was purified by flash chromatographyas in example 1a to give 28 mg of the title compound as a colorless oil.

IR: (CDCl₃, cm⁻¹) 2960 (m), 1724 (vs), 1426 (m), 1255 (vs);

MS: (DCl/NH₃) m/e 240 (M+NH₄);

NMR: (300 MHz, CDCl₃) ∂7.6-7.3 (m, 5H, phenyl), 4.18 (B₂ of A₂ B₂, 2H,CH₂ O), 1.99 (s, 3H, Me), 1.25 (A₂ of A₂ B₂, 2H, CH₂ Si), 0.35 (s, 6H,SiMe);

¹³ C NMR: (75 MHz, CDCl₃) ∂171.1 (CO), 138 (ipso), 133.4 (meta), 129.2(para), 127.9 (ortho), 62.3 (CH₂ O), 21.1 (Me), 16.5 (CH₂ Si), -2.9(SiMe).

Elemental Analysis: Calc'd for C₁₂ H₁₈ O₂ Si; C: 64.82; H: 8.16. Found;C: 65.02; H: 8.07.

b) Preparation of 1,1-Dimethyl-1-phenyl-1-silapropane-3-ol

The remaining 8.29 g of crude product from part b, above, was worked upas in the case of example 1 to give 1.64 g of1,1-dimethyl-1-phenyl-1-silapropane-3-ol as a colorless oil, 23%overall.

IR: (CDCl₃, cm⁻¹) 3616 (m), 2960 (m), 1425 (m), 1251 (s);

MS: (DCl/NH₃) m/e 198 (M+NH₄);

NMR: (300 MHz, CDCl₃) ∂7.6-7.3 (m, 5H, phenyl), 3.75 (B₂ of A₂ B₂, 2H,CH₂ O), 1.49 (s, 1.2H, OH), 1.22 (A₂ of A₂ B₂, 2H, CH₂ Si), 0.33 (s, 6H,SiMe);

¹³ C NMR: (75 MHz, CDCl₃) ∂138.5 (ipso), 133.4 (meta), 129 (para), 127.8(ortho), 59.9 (CH₂ O), 21.1 (CH₂ Si), -2.8 (SiMe).

Elemental Analysis: Calc'd for C₁₀ H₁₆ OSi.0.1 H₂ O; C: 65.92; H: 8.99.Found; C: 65.95; H: 8.97.

EXAMPLE 3 a) Preparation of 1,1,1-Triethyl-3-acetoxy-1-silapropane

To a solution of 6.39 mL (40 mmol) of Et₃ SiH and 3.69 mL (40 mmol) ofvinyl acetate in 40 mL of toluene is added 61.3 mg (0.16 mmol) of Rh₂Cl₂ (CO)₄. Caution: the reaction evolves heat and gas. Within about 5min, the reaction mixture darkens in color. Reaction is judged completeby TLC analysis (10% EtOAc in cyclohexane) after 1 h. The reaction isworked up and purified, if desired, as in example 1a.

b) Preparation of 1,1,1-Triethyl-1-silapropane-3-ol

The crude product from part a) can be worked up as in the case oftriphenylsilylethanol (example 1) to give the1,1,1-triethyl-1-silapropane-3-ol.

EXAMPLE 4 Preparation of 1,1,1-Triphenyl-1-silaheptane-7-ol (5)##STR14##

The acetate of 5-hexen-1-ol is prepared by refluxing 4.8 mL (40 mmol) ofthe alcohol in 15/15 mL of pyridine/acetic anhydride for 4 h. Thesolvents are removed in vacuo, and the residue is thoroughly vacuumdried. The crude acetate is dissolved in 40 mL of toluene, and 10.42 gof triphenylsilane is added, followed by 77.8 mg (0.25 mmol) of Rh₂ Cl₂(CO)₄. The reaction is stirred at room temperature under N₂ for 24 h,during which time the reaction turns dark brown in color. Somequantities of the isomer 2-methyl-1,1,1 triphenylsilahexan-6-ol can beexpected. If necessary, the isomers can be separated by chromatography.Workup as in example 1a, followed by base hydrolysis as in 1b, affordsthe title compound, (5).

B. Preparation of Phosphorylating Reagents EXAMPLE 5 Preparation of2-Trimethylsilylethyl-2-cyanoethyl-N,N-diisopropylaminophosphoramidite(1)

To a solution of 573 μL (4 mmol) of 2-trimethylsilylethanol(commercially available from Aldrich Chemical, Milwaukee, Wis.; orprepared in a manner analagous to example 2, above) and 1.39 mL (8 mmol)of i-Pr₂ NEt in 8 mL of THF at 0° C. was added 892 μL (4 mmol) of2-cyanoethyl-N,N-diisopropylaminochlorophosphoramidite all at once. Thereaction became very cloudy almost immediately. The ice bath wasremoved, and the reaction stirred to room temperature overnight, for atotal of 19 h. After filtration to remove i-Pr₂ NEt-HCl, the THF wasevaporated. The residue was partitioned between 50/50 mL EtOAc/0.1M Na₂CO₃, pH 12. After phase separation and solvent removal of organic phase,the residue was vacuum dried. Flash chromatography using 12% EtOAc incyclohexane on a 150 mm×25 mm ID column afforded 573.8 mg (78%) of thetitle compound as a water-white viscous oil, R_(f) =0.65 in 15% EtOAc incyclohexane.

MS: (DCl, NH₃) 319 (M+H), 291 (M-HCN);

NMR: (CD₂ Cl₂) ∂3.9-3.62 (m, 4H), 3.56 (dsept, 2H, J_(CH) =7.0 Hz,J_(PH) =10.0 Hz, NH), 2.59 (t, 2H, J=6.2 Hz, CH₂ CN), 1.15 (dd, 12H,J_(CH) =7.0, J_(PH) =2.2 Hz, Me), 0.97 (tq, 2H, J=8.0, 0.7 Hz, CH₂ Si),0.03 (s, 9H, SiMe).

EXAMPLE 6 Preparation of2-Triphenylsilylethyl-2-cyanoethyl-N,N-diisopropylaminophosphoramidite(6) ##STR15##

To a solution of 3.04 g (10 mmol) of (4), 4.18 mL (24 mmol) of i-Pr₂NEt, and 5 mg of 4,4-dimethylaminopyridine in 15 mL of THF at 0° C. wasadded 2.68 mL (12 mmol) of2-cyanoethyl-N,N-diisopropylaminochlorophosphoramidite (5) all at once.A white precipitate formed almost immediately. Reaction was completeafter 30 min at 0° C. After solvent removal, the residue was partitionedbetween 100/100 mL 0.1M Na₂ CO₃ /EtOAc, and the phases separated. Theaqueous phase was re-extracted with 50 mL EtOAc, and the combinedorganic phases were concentrated and vacuum dried. Flash chromatography(10% EtOAc in cyclohexane) using a 41 mm I.D.×150 mm long silica gelcolumn gave 3.35 g of (6) (66%) after vacuum drying overnight as aviscous colorless oil. This material gradually crystallized in a -20° C.freezer over the course of several weeks. During the chromatography, 100μL NEt₃ was added to each fraction, in order to minimize the effects ofadventitious acid in the fraction tubes or in the silica gel used forflash chromatography.

IR: (film, cm⁻¹) 2962 (m), 1426 (m);

MS: (DCl/NH₃) m/e 505 (M+H);

NMR: (300 MHz, CD₃ CN) ∂7.6-7.3 (m, 15H, phenyl), 3.9-3.7 (m, 2H, CH₂O), 3.66 (dt, 2H, J_(CH) =5.9 Hz, J_(PH) =7.7 Hz, CH₂ O), 3.51 (dsept,2H, J_(CH) =6.6 Hz, J_(PH) =9.9 Hz, NH), 2.54 (t, 2H, J=5.5 Hz, CH₂ CN),1.87 (br t, 2H, J=6.3 Hz, CH₂ Si), 1.07 (dd, 12H, J_(CH) =6.6 HZ, J_(PH)=29.4 Hz, Me);

¹³ C NMR: (75 MHz, CD₃ CN) ∂136.3 (meta), 135.5 (ipso), 130.7 (para),129 (ortho), 117.7 (CN), 61.1 (d, J_(PC) =18.3 Hz, CH₂ O), 59.3 (d,J_(PC) =18.3 Hz, CH₂ O), 43.6 (d, J_(PC) =12.2 Hz, NCH), 24.8 (virtualt, J_(PC) =7.3 Hz, Me), 21 (d, J_(PC) =7.3 Hz, CH₂ CN), 17.2 (d, J_(PC)=7.3 Hz, CH₂ Si);

³¹ P NMR: (202 MHz, CD₃ CN) ∂145.6.

EXAMPLE 7 Preparation of2-Triethylsilylethyl-2-cyanoethyl-N,N-diisopropylaminophosphoramidite

Example 5 is repeated except the product of example 3b is used as thestarting compound to produce the title compound.

EXAMPLE 8 Preparation of2-bismethylphenylsilylethyl-2-cyanoethyl-N,N-diisopropylaminophosphoramidite

Example 6 is repeated except the product of example 2b is used as thestarting compound to produce the title compound.

EXAMPLE 9 Preparation of Triphenylsilylethyl H-phosphonate-DBU reagent

To a solution of N-methylmorpholine (89 equiv), triazole (33 equiv) andPCl₃ (10 equiv) is added triphenylsilylethanol at 0° C. The reaction isstirred at this temperature for 2.5 h. The reaction is then quenched byaddition of 100 mM 1,5-diazabicyclo [5.4.0] undec-5-ene(DBU)-bicarbonate, and the phases are separated. The organic phase isstripped to dryness in vacuo, and the crude H-phosphonate-DBU ispurified by chromatography.

C. Preparation of Phosphorylated, Protected Oligonucleotides andDeprotection Thereof EXAMPLE 10 a) Use of (6) in AutomatedPhosphorylation of DNA ##STR16##

The phosphoramidite (6) (example 6, above) was used to phosphorylate a25-mer oligonucleotide at the 1 μmol level using an ABl (Foster City,Calif.) 380A DNA Synthesizer. The phosphoramidite couplings were runusing the synthesis program from the manufacturer except that the "wait"time (time of contact of phosphoramidite solution with support) and"wash" time are both doubled. The preparative HPLC run, showingseparation of the failure sequences from full-length oligo, is shown inFIG. 1.

b) Deprotection of Phosphorylated Oligonucleotide ##STR17##

The collected material (7) from part a) was dried in vacuo, then ethanolprecipitated. The purified DNA was then desilylated using 100/100 μL ofDMSO/1.0M Tetra-n-butyl ammonium Fluoride (TBAF) (Aldrich, Milwaukee,Wis.). The reaction was performed in a 68° C. heating block for 3.5 h.The reaction was diluted to 500 μL with 300 μL of water, and thereaction was desalted by passage down a NAP-5 column (Pharmacia,Piscataway, N.J.). The 1.0 mL eluate was dried in vacuo, then wasethanol precipitated to give purified, terminally phosphorylated DNA.HPLC analysis of this material is shown in FIG. 2.

EXAMPLE 11

Example 10 is repeated except the phosphoramidite reagent of example 5is used in place of the phosphoramidite reagent of example 6.

EXAMPLE 12

Example 10 is repeated except the phosphoramidite reagent of example 7is used in place of the phosphoramidite reagent of example 6.

EXAMPLE 13

Example 10 is repeated except the phosphoramidite reagent of example 8is used in place of the phosphoramidite reagent of example 6.

EXAMPLE 14 Phosphorylation by the H-phosphonate method

The reagent from Example 9 is used to phosphorylate an oligonucleotideusing the general reaction protocol and conditions of Froehler, et al.,Tetrahedron Letters, 27:469-472 (1986) except the coupling reagent isadamantoyl chloride and the capping reagent is β-cyanoethyl hydrogenphosphonate. After adamantoyl chloride catalyzed coupling of the5'-hydroxyoligonucleotide with the triphenylsilylethyl hydrogenphosphonate is complete, all H-phosphonate linkages in theoligonucleotide are oxidized with iodine to the phosphodiester oxidationstate. The oligonucleotide obtained can be separated on HPLC in the samemanner as DNA of identical sequence which is prepared usingphosphoramidite chemistry. This material may be desilylated in the samefashion as the phosphoramidite-prepared oligonucleotide.

What is claimed is:
 1. A process for synthesis of silyl alcohols,comprising:a) hydrosilation of an vinylic ester of the formula ##STR18##wherein R_(a), is a straight or branched alkenyl group having from 2 toabout 20 carbons and R_(b) is H or lower alkyl, with a silane of theformula ##STR19## wherein R₅, R₆ and R₇ are independently selected fromthe group consisting of H, alkyl, aryl, substituted alkyl, substitutedaryl, oxa and thia analogs of alkyl, aryl, substituted alkyl andsubstituted aryl, and halogen, in the presence of a metal catalystcapable of catalyzing the addition of H and a silyl group across thedouble bond of the alkenyl group to form a silyl substituted ester; andb) hydrolyzing the silyl substituted ester to an alcohol in the presenceof mild base.
 2. The process of claim 1 wherein R_(a) is lower alkenyl.3. The process of claim 2 wherein R_(a) is vinyl.
 4. The process ofclaim 1 wherein R_(b) is lower alkyl.
 5. The process of claim 4 whereinR_(b) is methyl.
 6. The process of claim 1 wherein at least one of R₅,R₆ and R₇ consists of a sterically bulky group.
 7. The process of claim6 wherein each of R₅, R₆ and R₇ consists of a sterically bulky group. 8.The process of claim 1 wherein at least one of R₅, R₆ and R₇ consists ofa hydrophobic group.
 9. The process of claim 8 wherein each of R₅, R₆and R₇ consists of a hydrophobic group.
 10. The process of claim 1wherein hydrosilation is done in a solution of toluene ranging fromabout 0.1 to about 4 molar.
 11. The process of claim 10 wherein saidsolution is about 1 molar.
 12. The process of claim 1 wherein said metalcatalyst is a transition metal complex having a metal selected from thegroup consisting of cobalt, nickel, platinum, palladium and rhodium. 13.The process of claim 12 wherein said metal catalyst is {RhCl(CO)₂ }₂.14. The process of claim 1 wherein said hydrolysis is done in a proticsolvent.
 15. The process of claim 1 wherein said mild base is analcoholic solution of K₂ CO₃.
 16. The process of claim 15 wherein saidK₂ CO₃ is present in base:acetate molar ratio of between 0.1:1 and2.5:1.
 17. The process of claim 14 wherein said protic solvent ismethanol.
 18. The process of claim 1, further comprising a step ofseparating the silyl substituted alcohol from undesired products of sidereactions.